Human studies suggest that retinol therapy in preterm infants ameliorates the morbid consequences of prematurity and conventional ventilation, which include alveolar simplification and subsequent chronic lung disease (CLD). Animal studies further reveal that retinol signaling regulates the processes that determine alveolar formation, but neither human nor animal studies have addressed the molecular mechanism by which either retinol amelioration or regulation occurs in CLD. Using a lamb model of prematurity, conventional ventilation, and subsequent alveolar simplification, we established that retinol therapy of conventionally ventilated preterm lambs (vitA+CV) permits alveolar formation that resembles the lungs of preterm lambs treated with continuous positive airway pressure (CPAP; preterm control), and term newborn lambs (gestation control). Furthermore, vitA+CV or CPAP treatment permits expression of surfactant protein B (SP-B), vascular endothelial growth factor (VEGF), and p53 (a marker of apoptosis) versus CV treatment. A major novel advancement by our group is that a specific RARa agonist stimulates normal alveolar formation in the face of conventional ventilation in our CLD model. Our focus on molecular mechanisms will be on vitamin A regulation of genes for SP-B (direct regulation), VEGF (indirect regulation), and p53 (indirect regulation). SP-B and VEGF are co-expressed by alveolar type II cells in the lung; p53 is expressed by mesenchyme. The overall hypothesis, tested by the following 3 specific aims, is that vitamin A therapy permits appropriate alveolar formation through the classical retinoid signaling pathway and results in both direct and indirect regulation of downstream gene products. Specific Aim 1 will determine the rescue of alveolar formation by vitamin A in chronically ventilated preterm lambs. Specific Aim 2 will compare CV with or without RAR/RXR agonists versus CPAP with or without RAR/RXR antagonists to identify mechanisms of action of vitamin A. This aim will test the hypothesis that vitamin A signaling in our model occurs through RAR(/RXR. Specific Aim 3 will identify the molecular mechanisms by which vitamin A impacts SP-B, VEGF, and p53 promoter function. We will use lung cells isolated from lambs to recapitulate the in vivo findings, and transient transfection of stable cell lines to isolate specific mechanisms. We will test the hypothesis that the promoter regions of our 3 target genes are retinoid responsive. Thus, this project will provide novel mechanistic insights about alveolar formation and its dysregulation in CLD.